BIOACTIVITY ASSESMENT OF
β
-TCP/ALGINATE
COMPOSITE FOR PRESERVATION ALVEOLAR RIDGE IN
SBF SOLUTION AND SHEEP AS ANIMAL MODEL
LIZA MARYETI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY BOGOR
STATEMENT OF THESIS
I, Liza Maryeti, hereby stated that this thesis entitled “Bioactivity
Assessment of β-TCP/alginate Composite for Preservation Alveolar Ridge in SBF solution and Sheep as Animal Model” is true of my own work under the supervisor advisory board and that it has not been submitted before in any form to any university. The content of this thesis has been examined by the advising advisory board and external examiner. Sources of information which is derived or cited either from published or unpublished scientific paper from other writers have mentioned in the script and listed in the References at the end part of this thesis.
I hereby assign the copyright of my thesis to Bogor Agricultural University.
Bogor, Mei 2016
Liza Maryeti
RINGKASAN
LIZA MARYETI. Analisis Bioactivity dari scaffold komposit β-TCP/alginate sebaagai pelestarian tulang alveolar pada larutan SBF dan domba sebagai hewan uji. Supervised by KIAGUS DAHLAN, GUNANTI dan YOSHIKI MATSUMOTO.
Resopsi tulang alveolar adalah proses fisiologi yang umum terjadi setelah kehilangan gigi. Pelestarian tulang alveolar setelah pencabutan gigi menjadi perhatian yang penting dalam kedokteran gigi sebelum penempatan implant. Penggunaan implant tulang sebagai pengisi pada soket alveolar setelah pencabutan gigi sangat disarankan untuk mencegah resopsi tulang dan membangun arsitektur tulang yang baik untuk penempatan implant. Dalam penelitian ini kami menggunakan scaffold betha tricalcium fospat dikombinasikan dengan alginate sebagai matriks untuk pelestraian dimensi alveolar ridge setelah proses kehilangan gigi. Penelitian ini bertujuan untuk mengetahui laju degradasi
dari scaffold β–TCP/alginate dalam larutan SBF dengan variasi waktu yaitu selama 0 sampai 90 hari perendaman. Utuk mengetahui sifat biokompatibiltas dari scaffold digunakan domba sebagai hewan uji.
Laju degradasi scaffold dalam larutan SBF menunjukkan penurunan berat sampel selama waktu perendaman. Hal ini mengindikasikan pertumbuhan apatite yang mengacu pada component tulang karena presipitasi ion Ca dan PO dalam larutan SBF. Selain itu, pelepasan calcium dan phosphate dari komposit juga di ukur dalam penelitian ini, dimana terjadi penurunan ion calcium.
Proses penyembuhan tulang pada soket setelah pencabutan gigi juga diamati pada hari ke 90 pasca operasi. Terlihat adanya pertumbuhan tulang baru berupa woven bone pada kelompok control dan kelopmok yang di beri implan. Data radiografi menunjukan hanya sedikit perubahan pada dimensi mesiodistal di daerah edontulus pada kelompok yang di beri perlakuan. Data histologi dan histomorphometry juga mengindikasikan presentasi kehadiran tulang baru yang lebih besar pada socket yang di isi dengan scaffold β–TCP/alginate (78%) dibandingkan dengan control (31%). Selain itu, kehadiran osteoid yang di deteksi dengan pewarnaan azan lebih banyak pada kelompok yang di isi dengan scaffold daripada yang dibiarkan kosong. Hal ini membuktikan sifat osteoinductivity dari
scaffold komposit β–TCP/alginate. Hasil penelitian ini menunjukkan bahwa
scaffold komposit β–TCP/alginate bisa digunakan sebagai pelestarian dimensi alveolar ridge.
SUMMARY
LIZA MARYETI. Bioactivity assessment of scaffold β-TCP/alginate composite for preservation alveolar ridge in SBF solution sheeps as animal model. Supervised by KIAGUS DAHLAN, GUNANTI and YOSHIKI MATSUMOTO.
Bone resorption is a physiological process after tooth extraction. Preservation of alveolar bone following tooth extraction is among the important goals in dental practices before dental implant placement. The use of bone substitutes to fill the tooth socket is suggested to prevent bone resorption and establish good bone architecture for implant placement. In this study we used scaffold beta-tricalcium phosphate (ß-TCP) combine with alginate for preservation alveolar ridge after extraction. This investigation purpose was to study degradation of scaffold β–TCP/alginate in simulated body fluid (SBF) solution and biocompatibility of scaffold in animal model.
Degradation rate with various immersing times for 0-90 days were conducted. This result showed a decrease in weight of sample during different time. This is indicates of growth the apatite who is a constituent component of bone, that is occur because of the precipitation of Ca and PO4 in the SBF solution. Furthermore, a continuous release of calcium and phosphate from the composite was measured, whereas in SBF, decrease of the amount of the two ions in the solution was observed accompanied with the formation of a CaP layer on the surface.
The extraction socket healing process is considered complete (90d) when the dental socket is filled by woven bone; it being the expression of mature bone markers prevalent at this period. The x-ray radiograph of sheep’s incisor indicated small change on mesio-distal of the edentulous area. Histological and histomorphometric confirms the area of new bone formation higher percentage in treatment than control. Histomorphometric analysis of the alveolar bone showed that it contained 78% new bone formation in extraction socket, and 31% new bone in socket left empty. In addition, the abundant of osteoid cell in socket filled with
β-TCP/Alginate was the proof of osteoinductivity of the composite. It was
obvious that the β-TCP/alginate scaffold composite could preservation alveolar ridge dimension on sheep.
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Copyright reserved by the law
Forbidden to quote part or all of these writings without including or mentioning the source. Citing is only for educational purposes, research, writing papers, drafting reports, writing criticism, or review an issue, and citing it does not harm the interests of fair Bogor Agricultural University.
A Thesis submitted in partial fulfillment of the requirement for Master Degree
in Biophysics Program
BIOACTIVITY ASSESMENT OF
β
-TCP/ALGINATE
COMPOSITE FOR PRESERVATION ALVEOLAR RIDGE IN
SBF SOLUTION AND SHEEP AS ANIMAL MODEL
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
Thesis title : Bioactivity Assesment of Scaffold β-TCP/aginate Composite for Preservation Alveolar Ridge in SBF Solution and Sheep As Animal Model
Name : Liza Maryeti
ID : G751130211
Approved by
The Commission of Supervisors
Dr Kiagus Dahlan Supervisor
Dr drh Gunanti, MS Co-Supervisor
Yoshiki Matsumoto, PhD Co-Supervisor
Certified by:
Head of Biophysics Graduate Program
Dr Mersi Kurniati, M.Si
Dean of the IPB Graduate School
Dr Ir Dahrul Syah, MSc Agr
Examination Date : May 20, 2016
FOREWORD
First and foremost, I would humbly distinguish the Most Gracious Allah SWT, all praises to Allah for the gifts and His blessing in completing this thesis
with title Bioactivity Assessment of Scaffold β-TCP/Alginate Composite for Preservation Alveolar Ridge in SBF Solution and Sheeps as Animal Model. This thesis submitted for the Degree Programs of Master Science in Master of Science of Biophysics. I would like to sincerely deliver my greatest gratitude to my advisor: Dr. Ir. Kiagus Dahlan, M.Si, Dr. drh. Gunanti, MS, Matsumoto Yoshiki, Ph.D for their advice, expertness, encouragement, and support. I thank to Dr. Akhirudin Maddu, M.Si as my examiner and representative of Biophysics program for the suggestion, Dr. Toni Sumariada who give me inspiration, and Mr. Rustami, M.Si for the useful discussion. I also thanks to my best friend Jayanti Dwi Hamdila, Fitri Afriani, Marliani, and Ibu Eli Aisah Sugiarti, for our togetherness about 3 years in Bogor, and thank a lot to my partner in crime Wahyu Kumala Sari for always listening and supporting me. So many thankful to Nur Aisyah Nuzulia and all friends in 2013’s Biophysics. To all of these people, I
owe its whole-hearted gratitude that impossible to describe.
My appreciations were also extended to Japan Student Services Organization (JASSO) Scholarship for granting scholarship during the study and experiment in Kagawa University and also for SUIJI-JDP (Six University Initiative Japan-Indonesia Joint Degree Program) who allowed me to expand my knowledge and experiences in Japan. This master thesis would not have been possible unless the funding of Indonesian Government scholarship (Beasiswa Freshgraduate). So many thankful to my all lab member (nao, kunikata, tagaki, imade ect) animal science, Kagawa University Japan, for unforgettable moment and precious togetherness and I also thank to all SUIJI-JDP student (UGM-IPB-UNHAS).
I would like to take this moment to deeply express my thankful feeling to my family Ibu, Ayah (alm), Uni, Abg, Niwit, Rezki and all my cousin and whole families members who have been praying, loving and supporting as always. I give all respect that impossible to describe, thank you so much. Finally, I hope this thesis can give information about advanced biomaterial from natural source. Nonetheless, I also welcome any critical feedback and advice from readers in order to maintain it as successful project. I do hope this thesis could be useful.
Bogor, Mei 2016
TABLE OF CONTENS
TABLE LIST ii
FIGURE LIST iii
APPENDICES LIST iv
1 INTRODUCTION 1
Background 1
Objective 2
Benefit 3
2 MATERIALS AND METHODS 3
Place and Time Schedule 3
Materials and Equipments 3
Experimental Method 4
Preparation of SBF 4
Animal and teeth 4
Surgical and grafting Procedure 4
Radiographic observation 4
Histological and Histomorphometric analysis 4
3 RESULTS AND DISCUSSION 5 Characterization of β-TCP/alginate composite scaffold 5 Biodegradation Test in SBF solution 5
Biocompatibility Test on Sheep 8
Radiographic Evaluation 8
Histological and Histomorphometry Analysis 10
4 CONCLUSION AND SUGGESTION 27
Conclusion 27
Suggestion 27
REFERENCES 27
APPENDICES 31
TABLE LIST
1 Animal codes
2 Mass of β-TCP/Alginate scaffold after different degradation time in SBF solution
3 Weight loss versus degradation time for the materials studied 7 The weight loss curve of scaffold has also been included to
4 illustrate its degradation behavior
5 Concentration ion Ca2+ and PO4 in SBF solution 8
9 Macroscopic results of sheeps mandible at Day+90 harvesting 10 (a) control (b) A1 (c) A2 (d) A3
10 Schema picture of alveolar ridge augmentation; untreated (a) 11 treated with β-TCP/Alginate
11 H&E staining of the extraction sockets following 3 month 12 treatments: (a) control group: new bone formation (*) mainly
composed of connective tissue (arrow); (b) filling with
β-TCP/alginate scaffold (A1): new bone formation (*) almost completely filled by compact bone tissue,
arrows show connective tissue; (c) filling with β-TCP/alginate scaffold
(A2): mostly new bone tissue (d) filling with β-TCP/alginate scaffold (A3); Specimens exhibited particles of involved by a thin calcified tissue, and fissures were observed in the particles (arrows), while the central portion showed connective cells (arrows). HE staining.
Magnification: 40x, Scale bar 100μm
12 Histomhorphometric showing the new one formation for control 13 and treatment (Using WinRoof Software)
14 MALDI-TOF MS imaging of alveolar bone, expressed localization of 15 optical image, upper : (a) control (b) treatment;
localization of collagen type 1 under (c) control (d) teatment
15 Histomhorphometric showing the area of collagen type 1 for 16 control and treatment (Using WinRoof Software)
APPENDIX LIST
1. Flow chart of the research
2. SBF result of Scaffold at D+7 PO 3. SBF result of Scaffold at D+30 PO 4. SBF result of Scaffold at D+60 PO
1 INTRODUCTION
Background
The most common dental diseases are periodontal disease, feline odontoclastic resorptive lesions and feline chronic gingivostomatitis. These diseases often cause the loss of teeth or require dental extraction. Alveolar bone loss can occur after tooth extraction, as a result of advanced periodontal disease or failed endodontic therapy. The resorption and remodeling of the alveolar ridge after tooth removal is a natural healing phenomenon, which is physiologically undesirable and possibly inevitable and can negatively impact implant placement (Zeeshan et al. 2015). If the alveolar ridge is not preserved at the time of tooth extraction or loss, alveolar ridge height and width may be lost, particularly in the area of the facial plate. Several system reviews have reported losses between 3 & 6 mm horizontally and 2 mm vertically (Araujo et al. 2005). Reduction of bone in the horizontal socket dimension of approximately 50% takes place over 1 year of healing. The early resorption of buccal bundle bone, which takes place during the first 8 weeks following extraction, proceeds with a marked reduction predominantly in the horizontal dimension (Mahmoud-A et al. 2013). A reduction in vertical ridge height of 0.8 mm over a 3 month period also predominates on the buccal aspect. Adequate volumes of alveolar bone which are close to the original dimensions of the alveolar process are necessary to provide favorable esthetics and successful long-term outcomes for dental implants (Phunke et al. 2012). Therefore, preservation of extraction socket dimensions has been attempted by many investigators immediately following tooth extraction.
Conventional tissue replacements, such as autografts (i.e.,the patient’s own
bone, which requires multiple and potentially painful procedures), allografts (i.e.,human bone, not from the patient), and xenografts (i.e.,animal bone) cannot meet the quantity and performance needed by the patients. Advances in biomaterials research and development of new and improved surgical techniques and armamentarium have resulted in an ever increasing use of dental implants for tooth replacement. A large number of 3-dimensional (3D) porous scaffolds have been developed to overcome traditional limitations and have been applied to repair bone defects. However, there are still many problems that need to be resolved to meet clinical requirements (Zeeshaan et al. 2015). Bone is a complex tissue mainly composed of nonstoichiometric hydroxyapatite [Ca10(PO4)6(OH)2]
and collagen (Turek et al. 1985). Approximately 30–35% of dry bone is of organic materials, 95% of which is type I collagen. It has been widely used as a skin substitute material. As the main inorganic component of bone, β-TCP has been widely used in many orthopedic and dental implant materials because of its bioactive, osteoconductive, and osteoinductive properties (Keilman et al. 2000).
2
accepted as a bioactive scaffold material for guided bone regeneration. In addition to the requirements for chemical composition of the scaffold material, an interconnected porous structure is necessary to allow cell attachment, proliferation, and differentiation, and to provide pathways for biofluids (Hyong-ho et al. 2012). Cell scaffolds provide the initial structural support and retain cells in the defective area for cell growth, metabolism and matrix production, thus playing an important role during the development of engineered tissues. Therefore, much attention is focused on porous composites of β-TCP and biodegradable polymers, such as, polylactic acid, gelatin, and chitosan and alginate (Lee et al. 2001).
Alginate is a biocompatible, hydrophilic, and biodegradable anionic polymer under normal physiological conditions and is widely used as an instant gel for bone tissue engineering (Hyon-ho et al. 2012). It has been studied as a useful biomaterial in diverse tissue engineering applications because of its hydrophilic surface promoting cell adhesion, proliferation and differentiation, good biocompatibility and good host response, high biochemical significance in hemostasis, angiogenesis and macrophage activation, biodegradability by lysozyme and other enzymes, bactericidal/bacteriostatic activity, and capacity to maintain a predefined shape after cross-linking (Bose et al. 2012). Therefore, alginate appears to be a very promising candidate for building a bone engineering scaffold as a natural 3D porous matrix.
Prior to the clinical use, biocompatibility and mechanical stability of new materials should be test under both initial in vitro and in vivo conditions (Nuzulia 2014). In vitro testing is used primarily as a first stage test for obtaining a bioactive scaffold for bone tissue engineering. Biomimetic mineralization is a process by which organisms form minerals in a bioenvironmental acute toxicity and cytocompatibility. Simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma has been proposed by Kokubo with the purpose of identifying a material with in vivo bone bioactivity instead of using animal for the experiments. Recently, it has been used as a biomimetic mineralization method to prepare biomaterials. While in vivo testing is used to demonstrate the tissue response to materials (Arora et al. 2011).
Objectives
The objectives of this study was to evaluate the bioactivity, biocompatible and biodegradable characteristics of β-TCP/Alginate composite material as a tooth preservation in SBF solution and sheep as animal models. There have been no
reports on the use of β-TCP combined with alginate for post extraction socket preservation.
Benefits
3
2 MATERIALS AND METHOD
Place and Time Schedule
This research was conducted from July 2014 through March 2016 which took place in Biophysics Laboratory-IPB, Faculty of Veterinary Medicine-IPB and Faculty of Agriculture-Kagawa University Japan.
Material and Equipment’s
The β-TCP/Alginate scaffold composite was prepared by Fitri Afriani from department of Biophysics-IPB with precipitation method. The ratio of β-TCP to Alginate were 70/30. Materials and equipment were used for degradation rate were NaCl, NaHCO2, KCl, K2HPO43H2O, MgCl2.6H2O, HCl 1M, CaCl2, Na2SO4,
(CH2OH)2CNH2, and AAS spectroscopy. While materials and equipment’s used
for biocompatibility testing were sheep as animal model, minor surgery set, dental surgery tools, anesthetic material, and surgery room for aseptical insertion. Then, materials used for histopathological examination were formalin 10%, nitric acid 5%, aquadest, aquabidest, alcohol, xylol, Hematoxilin-Eosin (HE), azocarmine G or aniline blue, sodium citrate buffer, EDTA, alcohol.
Experimental Method
Preparation of SBF
Simulated body fluid (SBF) with ion concentration nearly equal to those of human blood plasma has been proposed by Kokubo with the purpose of identifying a material instead of using animal for the experiments (Kokubo et al.
2002). SBF was prepared in accordance with Kokubo’s method. The ion concentration (mM) are as follows: 7,996 gr NaCl; 0,350 gr NaHCO2; 0,224 gr
KCl; 0,228 gr K2HPO43H2O; 0,305 gr MgCl2.6H2O; 40 mL HCl 1M; 0,278 gr
CaCl2; 0,071 gr Na2SO4; and 6,057 gr (CH2OH)2CNH2. Samples were placed in a
shaking waterbath at 37 °C for a maximum of three months. Calcium and phosphate concentration were analyzed after 7, 30, 60 and 90 days. Calcium and phosphate concentration were determined using AAS spectroscopy. For degradation tests, the scaffold were accurately weighed before and after immersion in SBF. The weight loss (WL) was calculated through Equation 1.
WL = (1)
where W0 is the initial weight of the specimen and W is the weight of the
specimen dried after different degradation times (7, 30, 60, 90 days). Animal and teeth
4
and water ad libitum. All experiments were conducted along the institutional guidelines for the care and use of laboratory animals. Prior to surgery, the animal models were observed closely for a week in order to check their health status. They were maintained under identical environment, management and standard diet with ad libitum supply of drinking water.
Table 1 Animal code conditions, the lower incisor was extracted and immediately after tooth extraction, the alveolar socket was filled with synthesized β-TCP/Alginate composites (height 1.6 cm, diameter 1 cm) that were sterilized initially by exposure to
ultraviolet light. As control requirement, one sheep’s lower incisor were extracted
in the same manner and unfilled with any tooth filler and the other sheep’s fill
with β-TCP/Alginate. Each surgery was performed under same veterinary surgeon. Then, the sheep were housed under a climate-controlled environment in stall of animal used of FVM Bogor Agricultural University.
Radiographic observation
The alveolar sockets that filled with β-TCP/Alginate tooth filler and unfilled with any tooth filler were monitored using a set of x-ray radiographic apparatus in day D+ 0 pre operation, D+0 post operation (PO), D+7 PO and D+30 PO, D+60, and D+90 PO for observing the alveolar bone healing on sheep. Histological and Histomorphometric analysis
5 The histological examination was done for observing the degree of new bone formation and the number of osteoid cell on alveolar ridge after extraction. Sections were examined with (10x magnification) using Olympus CX20 microscope (Olympus, Japan) attached to a camera and computer. All the stained sections were examined by image analyzer computer system using the Image software (NIH, version v1.45e, Japan) capable of performing high speed digital image processing for the purpose of tissue measurements. Image software was calibrated and the images were opened on the computer screen for pre-analysis adjustments. For histomorphometric analysis of the area percent of bone, the color of bone trabecular was automatically selected, converted into grey then masked by a red color which allowed automatic measurement by the computer system using WinRoof Software version 7.4 (Japan).
3 RESULT AND DISCUSSION
Characterization of scaffold β-TCP/Alginate composite
Pore structure is an essential consideration in the development of scaffolds for tissue engineering. Pores must be interconnected to allow for cell growth, migration and nutrient flow. If pores are too small cell migration is limited, resulting in the formation of a cellular capsule around the edges of the scaffold (Murphy et al. 2009). Scaffolds with mean pore sizes ranging from 20 mm to 150 mm have been used in bone tissue engineering applications. Kalfas (2007) reported, the pore sizes are good for bone growth ranged between 200-400 µm. Pores allow for cell infiltration, tissue growth and facilitate the formation of new cells. Therefore, samples were used in this study has pores in the range of 150-300 µm and porosity 67%.
Figure 1a demonstrates the microstructure of the porous β-TCP/Alginate (pore size 150-300 µm). The appearance of β-TCP/alginate scaffold was a block mass (Figure 1b).
(a) (b)
6
Biodegradation Test in SBF solution
Simulated body fluid (SBF) is a solution with ion concentration and pH value similar to that of human blood plasma. SBF is known to cause the production of bioactive calcium phosphate precipitation similar to biological mineralization. The purpose for the use of SBF was to simulate human physiological condition. Human body fluid is supersaturated with respect to biological apatite, which constitutes the mineral phase of calcified tissue such as bone, dentine, and enamel in the body and also some pathological calcifications (Alonso et al. 2012).
In this experiment mass change was used to determine apatite formation or
degradation of β-TCP/Alginate. The data collected was the mass change of the surface or inside the sample; when this happens the apatite formation has a greater effect on the weight property of the ceramic than does the dissolution effect. As expected, the samples that showed a decreased in mass (Fig. 2) also showed apatite formation on the surface and inside the samples.
Table 2 Mass of β-TCP/Alginate scaffold after different degradation time in SBF solution immersion in SBF over a period of 3 month.
7 Figure 2 shows the initial and final setting time of scaffold β -TCP/Alginate weight different time. Biodegradation processes of scaffold β -TCP/Alginate was 80 % weight loss from the beginning. Degradation is a critical parameter of biomaterials. In the case of bone graft substitutes, degradation rate of the material should be comparable to the rate of new bone formation, in order for the material to provide sufficient support while leaving space for tissue growth.
Figure 3 Weight loss versus degradation time for the materials was studied. The weight loss curve of scaffold has also been included to illustrate its degradation behavior.
Yuliana (2015) reported that degradation of sample β-TCP alone the initial and final setting times were lower than scaffold β-TCP/Alginate by 10% on times experiments days 15, while in this study the scaffold β-TCP/Alginate degradation by 20% over 7 days (Figure 3). This phenomena on that cells immersion into SBF solution could remarkably slow down. The reason may be explained in this way, early bone formation and mineralization deposited could cover the inner surface which might have limited its exposure to surrounding body fluid. This might have prevented the implanted grafts exposed to solution, slowed down of material degradation. Another explanation might be associated with mechanical forces. Navaro et al reported that β-TCP was hardly resorbed in a non-loading calvarias model, while it could be easily degraded in the load-bearing area as other researchers observed (Zhao et al. 2011; Shahabooi et al.2015)
This results indicates that scaffold β-TCP/Alginate more rapidly degraded because β-TCP has similar composition with bone and in addition to the pore, adding alginate also affect the rate of degradation. On the other hand, release of calcium and phosphate from β-TCP/Alginate scaffold was measured in SBF solution over the time degradation of this study.
8
Figure 4 Concentration ion Ca2+ and PO4 in SBF solution over a period
of 3 month. It could be seen, increase of ion calcium concentration and decrease of ion phosphate concentration in SBF solution during period time.
Figure 4 shows the release of calcium and phosphate from the β -TCP/Alginate composite was measured in SBF solutions over the time of the degradation study. It could be seen from the Figure 4, when ion calcium concentration increase, the concentration of phosphate was decrease. It might be caused by the release of ion calcium from the scaffold could absorbed the ion phosphate from solution to scaffold. β-TCP/Alginate exhibited little ability to induce calcium phosphate apatite formation on its surface; Xin has also reported
similar trends with β-TCP in SBF (Zhao et al. 2011). After 90 days some of the pores were filled with apatite. The apatite formation on the inside of the sample may have been due to the local release of calcium and phosphorous in the pores which may be more favorable to promote apatite growth in the enclosed environment rather than the free surface. Release of calcium from a Ca-P ceramic is accompanied by other events, such as phosphate release, reprecipitation of a biological apatite layer, possibly containing endogenous proteins and other factors, and change of surface topography, all of which can affect bioactivity of the material as well. We can conclude that degradation was actively occurring in the
β-TCP/Alginate compoiste over the 90 days SBF treatment. Radiographic Evaluation
There were no clinical complications and the extraction wounds healed uneventfully. Studies have shown long-term successful osseointegrated implant with the satisfactory esthetic outcome can be achieved when applied with alveolar ridge preservation after extraction. Whereas, studies conducted in the past by various authors have also shown that the socket without graft has significant resorption rate in both vertical as well as buccolingual dimensions. X-ray images were taken under general anesthesia at 7, 30, 60, 90 days postoperation to follow
9 up the scaffold degradation as well as the new bone formation. Radiographic evidence of scaffold resorption as well as new bone formation was highly variable among the socket left empty and socket fill with β-TCP/Alginate.
(a) (b)
Figure 5 The radiograph of dorsoventral (DV) position of sheep ’s jaw pre- Operation (Scale bar 15mm).
The radiograph of sheeps jaw pre operation was shows in Figure 5a as control and figure 5b as treatment. This demonstrate the alveolar bone resorption that could be seen from the narrower mesiodistal of edentulous are along the observing time. As seen in Figure 6a, there was a larger distance of the alveolar ridge after tooth extraction due to the unfilled socket. The radiopacity in control could be visualized easily at 7 days after implantation (Fig. 6b), but it decreased dramatically overtime due to scaffold degradation. The dimension of the alveolar ridge had changed at D+7 PO (Figure 6b) and there is no significant change at D+30 (Figure 6c), but clearly followed by significant change at D+60 (Figure 6c) and D+90 PO (Figure 6e). Nevertheless, most of the β-TCP/Alginate implants had been resorbed at 90 days post-operation with a lower radiopacity and minor augmented alveolar ridge remained.
(a) (b) (c) (d) (e) Figure 6 The radiograph of dorsoventral (DV) position of sheep’s jaw of control
(unfilled with sample). There was large mesio-distal and low opacity on
the extraction site ( ) at (a) day+0 post operation (b) day+7 post
operation that become narrower at (c) day+30 post operation (d) day+60 post operation (e) and there was high alveolar bone resorption
10
In socket that filled with β-TCP/Alginate showed the expected goal. It was shown in Figure 7 that resubstituting process of β-TCP/Alginate and preservation on the alveolar ridge. The implant was radiopaque due to the property of β-TCP scaffold itself. However, the interface between the implant and the host bone began to show radiopacity indicating new bone formation and remodeling at this area. It was obvious from width of the edentulous area that has no significant difference. The same alveolar ridge width was observed at D+7 PO with β -TCP/Alginate resorption due to the decreasing of opacity on the alveolar socket (Figure 7b). There was a little change on alveolar ridge at D+60 PO as shows in Figure 7d. Certain degree of degradation was evident based on the appearance of radiotranslucent area inside the graft. The interface between the graft and the host bone remained radioopaque. At 90 days post-operation, radiopacity of elevated alveolar began to increase and the remodeling was obvious by bone texture, indicating newly formed trabecular bone, and at this time point augmented alveolar ridge maintained the original height, and the interfaces between the grafts and host bones were hard to distinguish.
(a) (b) (c) (d) (e) Figure 7 The radiograph of sheep (treatment) at (a) day+0 post operation (b)
day+7 post operation (c) day+30 post operation (d) day+60 post operation (e) day+90 post operation. The same width of mesio-distal indicated there was no alveolar bone resorption.
The detailed mesiodistal of the edentulous area is shows in Table 3. It is worth to note that β-TCP/Alginate as a tooth filler could stimulate the alveolar bone healing process and maintain the desired alveolar ridge. This preservation could be done before prostheses or implant placement.
11 To evaluate the effects of augmented alveolar bone, the width was measured. As the shows in Table 3, the augmented width decreased significantly at 90 days to only 5.7- 4.5 mm for control group. On the contrary, alveolar augmentation width remained 5.0-4.9 mm (A1), 5.8- 5.2 mm (A2) and 5.5-5.2 mm (A3). This width indicating the equivalent bone formation between the tissue engineered construct and the autologous bone implants
Macroscopic result of D+90 harvesting day confirms the bone healing process on postextraction socket shows in Figure 8. This funding was also consistent with the results reported by Nuzulia (2014), which observed a reduction in the early (30 days) phase of healing.
Biocompatibility test was also done in sheep to observe the alveolar bone healing process by resubstituting process of the corresponding β-TCP/Alginate. Histological examination showed formation of woven bone within the extraction socket. Areas of connective tissue were seen among bone trabeculae, indicating that the regeneration of extraction socket occurred by intramembranous ossification. The percentage of newly formed bone within the extraction socket was measured by the histophotometry. The general findings showed evidence of remodeling and new bone surrounding the graft fragments.
(a) (b)
12
(a) (b)
(
(c)
(d)
Figure 10 H&E staining of the extraction sockets following 3 month treatments: low magnifications image of extraction site (a) control group (b) treatment group (magnification: 40x, scale
bar 100μm). Representative high magnifications view (c) control group; new bone formation (*) mainly composed of connective tissue (arrow); (d) filling with β-TCP/alginate scaffold; new bone formation (*) almost completely filled by compact bone tissue. HE staining. Magnification: 100x, Scale
bar 100μm.
Figure 10 shows the area of woven bone in control group was lower than treatment group. The general findings showed evidence of remodeling and new bone surrounding the graft fragments. Three month after extraction of sheep lower incisor, the extraction socket was filled with trabeculae of cancellous bone. The result of histological examination of this study is in agreement with those obtained in experiments in which the observation period was extended by several weeks. Indovina Jr and Block evaluated the healing response with different bone substitute materials in extraction sockets in dogs. Histological examination made after an observation period of 8 weeks showed that the sockets left empty contained woven bone.
13
Figure 11 Histomhorphometric showing the new bone formation for control and treatment (Using WinRoof Software). The graph shows the
percentage of the area bone formation in treatment higher than control.
For histomorphometric analysis of the area percent of bone, the color of woven bone was automatically selected, converted into grey then masked by a red color which allowed automatic measurement by the computer system. The histomorphometric data of the present study showed that there was significant difference among groups, i.e.,the percentage of new bone tissue in extraction sockets filled with woven bone (78%) was different to that observed in sockets left empty (31%), three month after extraction. In the β-TCP alone as Nuzulia (2014) reported, however, the augmented alveolar ridge could have preserved dimension of alveolar ridge overtime but there is still residual material on the graft,
indicating that β-TCP alone has served only as an osteoconductivity scaffold and less biodegradable. This present study indicates that alginate has been shown to induce osteoblastic, differentiation and proliferation of bone marrow mesenchymal stem cells, could promote new bone formation and mineralization inside the elevated space at much early stage.
Β-TCP/Alginate works through both osteoinduction and osteoconduction. The material induces osteoblasts and chondroblasts differentiation from mesenchymal cells. With its osteoinductive properties alginate leads to an increase the number of available osteoblasts at the graft site (Wen sun et al. 2010). The
presence of β-TCP which supports osteoblasts adhesion (Bozidar et al. 2008), combined with alginate at the defect site, can explain the higher percentage of woven bone that was found in the treatment group.
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scaffold was actively generated, suggesting that degradation of β-TCP/Alginate promoted osteoid production (Navaro et al. 2004).
and treatment (Using WinRoof Software). This graph shows the number of osteosid cell in socket that fill with β-TCP/Alginate higher than socket left empty.
15 formation ingrowth of capillaries and bone), and (3) osteoconduction (i.e.,defined as the presence of differentiating factors that facilitate the recruitment and differentiation of mesenchymal stem cells and specifically induce them to form osteoblasts which deposit the new bone), in which the graft acts as a scaffold for deposition of new bone by adjacent living bone (Tuerky et al, 2000). The β-TCP combine with alginate has good biocompatibility and osteoconductive capacity (Charlene et al. 2014). Compared with other bone substitutes (e.g. PLGA scaffolds), β-TCP is characterized by its precisely defined physical and chemo-crystalline properties, high level of purity and uniformity of chemical composition, so that its biological reactions can be predicted reliably (Shahabooi et al.2015). It can be fabricated into high porosity scaffolds with good interconnectivity, which will ensure intercellular communication among osteogenic cells rested in lacunae (Dubravka et al. 2002). The macro-porosity of the material will facilitate cells adhesion and growth, and facilitate bone ingrowth and especially vascularization (Shahabooi et al.2015). These findings suggested that β-TCP/Alginate had served as a good scaffold for osteoblasts to increase the bone area and mineralization which has led the tissue-engineered complex to maintain the width of the augmented alveolar ridge throughout the experiment.
Yuan J et al. implanted porous β-TCP to repair canine mandibular bone segmental defects, and found that most of the material was degraded in load-bearing area 26 weeks post-operation. Lu J et al. has reported that 55% of β-TCP could be degraded after 24 weeks of implantation in a rabbit model of femoral condyle implantation (David t al. 2013). The degradation of β-TCP/Alginate in vivo is believed to involve in two pathways: solution-mediated dissolution and a cell-mediated resorption process (Middleton et al. 2000; David t al. 2013).
In this experiment, from the dramatically decreased width of augmented alveolar ridge based on X-radiograph images, biodegradation on SBF solution and general observations, we found that β-TCP/Alginate degraded rapidly.
Histological results demonstrating of β-TCP/Alginate are in accordance with those of other studies which have shown that the material can be replaced completely by new bone within a 3 months healing period. Results of this study demonstrated higher percentage of new bone formation and high number of osteoid cell. This is in agreement with the in vitro analysis of a previous study that showed that the rate of degradation of the graft material was proportional to the amount of new bone regeneration.
4 CONCLUSION AND SUGGESTION
Conclusion
In the present study, degradation of scaffold β-TCP/Alginate was analyzed during a three month period in SBF solution and bioactivity also done in sheeps as animal model. Degradation test in SBF solution showed that scaffold β -TCP/alginate more rapidly degraded because in addition of the pore, adding alginate also give effect to the rate of degradation. The degradation was actively
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Biocompatibility test of β-TCP/Alginate composite for preservation alveolar ridge post extraction shows a promising challenge in dental practices. The alveolar bone healing process is obviously observed on sheep showed by mesio-distal of the edentulous area that remains same alveolar ridge width and the presence of new bone formation proved by histological analysis. Histomorphometry results also show that this scaffold is biocompatible, attested by presence new bone and osteoid cell in treatment higher than the control. This indicates that β-TCP with calcium sources from egg shells combine with alginate potential as a tooth filler.
Suggestion
17 REFERENCES
Afriani, Fitri. 2015. Perancah Berpori dan Betha Tricalsium Phosphate dari limbah cangkang telur ayam dengan porogen alginate. [Thesis]. Bogor :
Faculty of Math and Natural Science : IPB.
Alonso L, Rasal G, Luis A. 2012. Development and characterization of α -tricalcium phosphate/monocalcium aluminate composite bone cement. J. Biomedical Science and Engineering. 5 :448-456.
Amler, M. H. 1980. The time sequence of tissue regeneration in human extraction wounds. Oral Surgery, Oral Medicine and Oral Pathology. 27 : 309– 318. Araoujo MG, Lindhe. 2005. Dimensional ridge alterations following tooth
extraction. An experimental study in the dog. J Clin Periodontol. 32 (20) : 212-218.
Arora N et al. 2011. Alveolar ridge preservation after dentalextraction and before implant placement:A literature review. Rev Odonto Cienc 26 : 77-83. Bozidar, Kovazdiz. 2008. Simple Preservation of a Maxillary Extraction Socket
Using Beta-tricalcium Phosphate with Type I Collagen: Preliminary Clinical and Histomorphometric Observations. JDCA. 74 : 66 – 74.
Bose F, Hamel L., Lerouxel E., Baslé M.F, Chappard D. 2005.Bone loss and teeth.
Joint Bone Spine. 72: 215–221.
Charlène B Danoux, Davide Barbieri, Huipin Yuan, Joost D de Bruijn, Clemens A van Blitterswijk. 2014. In vitro and in vivo bioactivity assessment of a polylactic acid/hydroxyapatite composite for bone regeneration. Biomatteral (12) : 31-40.
David L, Lian P. 2013. Soft and Hard Tissue Histologic Dimensions Around Dental Implants in the Canine Restored with Smaller-Diameter Abutments: A Paradigm Shift in Peri-implant Biology. The International Journal of Oral & Maxillofacial Implants. 28 (2): 494-502.
Dubravka K, Asja C. 2002. Resorptive Changes of Maxillary and Mandibular Bone Structures in Removable Denture Wearers. Acta Stomat Croat. 36(2) : 261-265.
Hoffmann, Oliver. 2008.Alveolar Bone Preservation in Extraction Sockets Using Resorbable dPTFE Membranes: A Retrospective Non-Randomized Study .J. Periodontal. 79) : 8 – 20.
Horowiz R et al. 2011. A Review on Alveolar Ridge Preservation Following Tooth Extraxtion. J Evid Base Dent Pract 31 (13): 149-160.
Hyeong-Ho J, Dong-Hyun, Tae W. 2012. In vivo evaluation of porous hydroxyapatite/chitosan–alginate composite scaffolds for bone tissue engineering. International Journal of Biological Macromolecules. 51(1) : 1079-1085.
Jayanti Y. 2015. Uji In vitro Hidroxyapatite dan β-Tricalcium Phospate dalam Simulated Body Fluid Sebagai Simulasi Biodegradasi Biomateria [Skripsi]. Kalfas F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M. 2012. Surgical protocols for ridge preservation after tooth extraction: a systematic review .Clin Oral Implants Res. 23( 5):22-38.
18
Kokovic V dan Todorovic L. 2011. Preimplantation filling of tooth socket with β- tricalcium phosphate/polylactic-polyglycolic acid (β-TCP/PLGA) root analogue: clinical and histological analysis in a patient. Vojnosanitetski Pregled. 34(4) : 366 -371
Lee, C.H., Singla, A., and Lee, Y. 2001. Biomedical applications of collagen. Int J Pharm 221 (1) : 31-40.
Mahmoud A, Hatem A. 2013. Evaluation of horizontal ridge augmentation using beta tricalcium phosphate and demineralized bone matrix: A comparative study. J Clin Exp Dent. 5(3) : 253-262.
Middleton JC, Tipton AJ. 2002. Synthetic biodegradable polymers as orthopedic devices. Biomaterials. 21(23):2335–2346.
Murphy KG. 1995. Postoperative healing complications associated with Gore-Tex periodontal material. Part I. Incidence and characterization. Int J Periodontics Restorative Dent. 15:363-75.
Navaro M, Ginebra M.P, Planell J.A. 2005. In vitro degradation behavior of a novel bioresorbable composite material based on PLA and a soluble CaP glass. Acta biomaterial. 1 : 411-419.
Nuzulia NU. 2014. Examination of chicken’s eggshell-basaed b tricalciumm
phosphate bioceramics as tooth filler”. [Thesis]. Bogor : Faculty of mathematich and natural sciences, IPB .
Phunke C, Zehlike T, Just T. 2011. Matrix change of bone grafting substitute after implantation into guinea pig bulla. Via medica journal. 71(2) : 109-114. Ravaglioli A et al. 2013. Mineral evolution of bone. Biomaterials. 19 ; 17:617-62
Sarah, Siti Aulia, “Alveolar ridge augmentation using distraction osteogenesis”.
World Journal Of Dentistry. (3): 174.
Sorensen WP, Loe H, Ramfjord SP. “Periodontal disease in the beagle dog: a cross sectional clinical study.” J Periodontal Res 1980; 15(4): 380-9
Shahabooei M, Razvi S. 2015. A histomorphometric study of the effect of doxycycline and erythromycin on bone formation in dental alveolar socket of rat. Advanced Biomedical Research.. 93 (2) : 115-121.
Simion M., Jovanovic S.A., Trisi P., Scarano A., Piattelli T. 1998. Vertical ridge augmentation around dental implants using a membrane techni que and autogenous bone or allografts in humans. Int. J. Periodontics Restorative Dent. 18 : 8–23.
Tan WL, Wong TL, Wong MC, Lang NP. 2012. A systematic review of post- extractional alveolar hard and soft tissue dimensional changes in humans (published online ahead of print Dec. 28, 2011). Clin Oral Implants Res
23( 5):1-21.
Tina M, Brian L. 2010. Histologic Analysis of Healing After Tooth Extraction With Ridge reservation Using Mineralized Human Bone Allograft.
Periodontal Journal. 81 (12) : 1765-1772.
Turek, S.L. 1985. Orthopaedics: Principles and Their Application, 2nd edition. Philadelphia: Lippincott Williams & Wilkins 1985, p. 113.
Tazaki J et al. 2009. BMP-2 release and dose response studies in hydroxyapatite and B- TCP. Bio- medical Materials and engineering. 19 ; 141 – 146. Turco G, Eleonora M, Bellomo F, Semeraro S, Donati I, Brun F, Grandolfo M,
19 Vignoletti F. 2009. Early healing of implants placed into fresh extraction sockets:
an experimental study in the beagle dog. J Clin Periodontol. 36 ; 265–277 Wang S, Zang Z. 2009. Vertical alveolar ridge augmentation with b-tricalcium
phosphate and autologous osteoblasts in canine mandible. Biomaterials. 30(2): (2489-2498).
Wen Sun, Weiwei Sun, Jingning Liu. 2010. Alterations in phosphorus, calcium and PTHrP ontribute to defects in dental and dental alveolar bone formation in calcium-sensing receptor-deficient mice. Development 137. 29 (5): 114-124.
Zeeshan S, Corneliu S. 2015. Bone Replacement Materials and Techniques Used for Achieving Vertical Alveolar Bone Augmentation. Materials. 8(2) : 2953-2993.
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21 Appendix 1 Flow chart of the research
In vitro in SBF solution In vivo test in sheeps
1. Degradation rate 2. Release ion Ca and PO4 3. Mass change
1. Radiography study 2. Biocompatibility test
3. Osteoconductivity potensial
Report arrangement Analysis data
Materials and equipment preparation
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BIOGRAPHY
Author was born in Sumatera Barat, Indonesia on March 13th, 1988. She is
the fourth daughter of Mr.Syaiful Azhar (Alm) and Mrs. Siti Hajir. She graduated from highschool MA Negeri 1 Pasaman 2007. She continued her study at Department of Physics, Faculty of Math and Natural Science, Padang State University in 2007 and officially she got her bachelor degree in 2011.
